The broad, long-term objective of this program is to study inositol metabolism in humans. Inositol is a key cellular metabolite. It is the precursor for the synthesis of phosphatidylinositol, an essential membrane lipid, an anchor for proteins, and a core component of the recently elucidated signal transduction mechanisms. The level of inositol biosynthesis appears to be tissue-specific, affected by hormones and some diseases. Aberrant concentrations of inositol have been implicated in neuropathy, vascular abnormalities and development of cataracts associated with diabetes. The aim of this project is the molecular genetic analysis of the regulation of inositol biosynthesis. Specifically, the project proposed is the isolation of a cDNA clone encoding the human inositol biosynthetic enzyme, inositol-l-phosphate synthase. The initial approach to cloning this gene will be the complementation of a yeast mutant strain, defective in this enzyme, with a clone bank of human cDNA in a yeast expression vector. Three alternate approaches which could be employed to isolate this clone are: 1) hybridization of the functionally analogous genes isolated from fungi (S. cerevisiae and C. albicans) to clone banks of human cDNA; 2) polymerase chain reaction using human mRNA as template and conserved regions of the fungal genes as primers, and; 3) purification of the human protein and subsequent generation of sequence information to synthesize precise probes for hybridization to clone banks and/or generation of antibody to screen expression banks. Once the clone has been obtained, as time permits, it will be characterized (restriction mapped and sequenced) to determine which regions of this protein have been conserved between organisms. This information should suggest which structural regions of the protein are essential for its functions. The cloned cDNA will be used to determine on which human chromosome the gene maps. In addition, the cloned cDNA will be used to examine tissue-specific expression of inositol-l-phosphate synthase. A direct consequence of this proposed research program will be attracting undergraduate students to the biomedical sciences, providing a modem research environment and training in molecular genetics and encouraging future graduate studies. This project is ideal in that it requires the use of many basic molecular biological and genetic techniques.